Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
  • B01D15/361—Ion-exchange
  • B01D15/362—Cation-exchange
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
  • B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

• the present invention relates to improved industrial scale processes for purifying metal cations, such as copper, from blood plasma derived albumin and pharmaceutical compositions derived therefrom.
• Serum albumin is one of the most abundant proteins present in blood (35-55 g/L; approximately 60% of total protein). It functions within the blood as a carrier of hydrophobic molecules such as fatty acids and as a scavenger that binds to a range of molecules including organic molecules and cations (such as Ca2+, Na+ and K+), sequestering such molecules until they can be eliminated. Because of albumin’s abundance, small relative size (66.5 kDa) and high charge at physiological pH its main function is to regulate the oncotic pressure of blood so as to maintain fluid in the bloodstream and prevent leakage into surrounding tissues
• hSA human serum albumin
• IV intravenous
• Albumin s unique physiochemical properties allow relatively effective purification by precipitation methods.
• Albumin has the highest solubility and the lowest isoelectric point (the pH at which it bears no net charge) of the major plasma proteins. Adjustments to pH, temperature, ionic strength, ethanol or salt concentration and protein concentration therefore allow the fractionation of albumin from the other plasma proteins.
• These fractionation methods include precipitation with cold ethanol, using methods such as Cohn or Kistler and Nitschmann for industrial scale manufacture of pharmaceutical grade hSA.
• cryosupernatant may be optionally exposed to an initial low ethanol (typically 8%) precipitation stage to remove Fibrinogen. Again, the precipitate (Fraction I) is removed and can be used to make other products such as Fibrinogen.
• Adsorption steps using ion-exchange resins or affinity are also optionally conducted across either of these two intermediate fractions to extract other proteins (e.g. Prothrombin complex; Antithrombin III; C1 esterase inhibitor).
• the albumin is extracted from the Supernatant I by raising the ethanol concentration to about 25% at about pH 6.9 for the Cohn method or about 19% at about pH 5.85 for the Kistler and Nitschmann method, the immunoglobulins are precipitated (Fraction (l+)ll+lll or Precipitate A) while the albumin remains in solution (Supernatant (l+)ll+lll or Filtrate A).
• the hSA is then isolated from the majority of the other plasma contaminants (mainly a and [3 globulins), which are precipitated by the further addition of ethanol to a final ethanol concentration of about 40% (Fraction IV).
• the albumin is itself precipitated near its isoelectric point.
• the precipitate paste (Fraction V) can be held frozen before further processing. It is important to recognise that these processes have some adaptability and have been optimised over the years to suit each manufacturers product portfolio. An example of this would be the presence or absence of an additional Cohn fractionation step (Fr IVi) following Fr (l+)ll+ll step that can be used to extract alpha-1 -antitrypsin.
• hSA utilised in the clinic is generally obtained by fractionation of pooled donor blood derived plasma.
• hSA manufacture is completed by solubilising the albumin, formulating with stabilisers (sodium caprylate and/or acetyl tryptophanate) and pasteurisation.
• the pasteurisation process 60 °C for 10 hours was introduced for albumin pharmaceutical products in the 1940s.
• the step inactivates lipid and non-lipid enveloped viruses including hepatitis A, B and C and HIV.
• the inclusion of the stabilisers ensures that the albumin solution is not denatured on heating.
• Most regulatory agencies require the step to be conducted in the final container, although terminal bulk pasteurisation has been accepted by a few regulatory agencies, such as the Therapeutic Goods Administration in Australia.
• hSA compositions are produced at two concentrations.
• the 4- 5% hSA solution is an isotonic solution particularly suitable for fluid replacement in hypovolaemia.
• the 20-25% hSA is a hypotonic but hyperoncotic solution for the treatment of fluid loss where electrolyte or fluid load is contraindicated.
• the highly concentrated protein solution provides colloidal pressure while minimizing the additional salts and fluid volume that are infused.
• the ideal pharmaceutical hSA product for these purposes would be monomeric albumin of very high purity, free from contamination with other plasma proteins, endotoxins, metal ions, albumin aggregates and prekallikrein activator (PKA).
• PKA prekallikrein activator
• these purification processes do not ensure the complete removal of contaminants, some of which may impact on the integrity of the final product and potentially influence the tolerability of hSA infusion.
• the present invention provides a process for reducing the level of metal cations in an albumin-comprising sample obtained from blood-derived plasma, the process comprising: i) providing an albumin-comprising sample obtained from blood derived- plasma; ii) contacting the albumin-comprising sample with a ligand for binding to or for sequestering metal cations to obtain a metal cation-depleted album incomprising sample; iii) recovering the metal cation-depleted albumin-comprising sample.
• the present invention provides a process for preparing a purified albumin composition from an albumin-comprising sample obtained from blood derived-plasma, the process comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma; ii) contacting the albumin-comprising sample with a ligand for binding to or for sequestering metal cations to obtain a metal cation-depleted album incomprising sample; thereby preparing a purified albumin composition.
• the ligand for binding to or for sequestering metal cations may be a chelating agent, or a cation exchange resin.
• the ligand for binding to or for sequestering metal cations is a chelating agent.
• the present invention provides a process for preparing a purified albumin composition from an albumin-comprising sample obtained from blood-derived plasma, the process comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma; ii) contacting the albumin-comprising sample with a chelating agent to obtain a metal cation-depleted albumin-comprising sample; thereby preparing a purified albumin composition.
• the chelating agent may be any agent which is capable of binding to and sequestering metal ions.
• the chelating agent binds to a divalent metal ion.
• the chelating agent binds to copper (II) ions, where the copper is optionally present in the albumin solution as copper (II) sulphide.
• a process for removing copper (cupric) ions from an albumin-comprising sample obtained from blood-derived plasma comprises: i) providing an albumin-comprising sample obtained from blood; ii) contacting the albumin-comprising sample with a chelating agent for binding to cupric ions in order to obtain an albumin-comprising sample that is depleted of cupric ions; thereby preparing a purified albumin composition.
• the chelating agent may be selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis([3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycinediacetic acid (MGDA), triethylenetetramine (Then), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tripolyphosphate (TPP), diethylenetriaminepentaacetic acid (DTPA), sodium diethyldithiocarbamate (DDC), L- Glutamic acid N,N-diacetic acid, tetrasodium salt (GLDA), and penicillamine, of any salt thereof, including a calcium or sodium salt thereof.
• EDTA ethylenediaminetetraacetic
• the chelating agent is a biodegradable chelating agent.
• the biodegradable chelating agent is selected from the group consisting of: ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycinediacetic acid (MGDA), and nitrilotriacetic acid (NTA), most preferably EDDS, or a salt thereof, including a calcium or sodium salt thereof.
• EDDS ethylenediamine-N,N'-disuccinic acid
• IDS iminodisuccinic acid
• MGDA methylglycinediacetic acid
• NDA nitrilotriacetic acid
• the biodegradable chelating agent is (S,S)-EDDS.
• the biodegradable chelating agent is the tri-sodium salt of (S,S)-EDDS.
• the chelating agent may be provided at any concentration that is suitable for depleting metal cations from the albumin-comprising sample, optionally wherein the chelating agent is contacted with the albumin-comprising sample at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the chelating agent is contacted with the albumin-comprising sample at a concentration of from about 2.5 mM to about 100 mM, preferably from about 5 mM to about 50 mM, or between about 10 mM to about 25 mM, more preferably from about 15 mM to about 20 mM.
• the concentration of albumin in the albumin-comprising sample is from about 40 g/L to about 250 g/L, and wherein the sample is contacted with chelator at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the concentration of chelator is from about 2.5 mM to about 100 mM, preferably from about 5 mM to about 50 mM, or between about 10 mM to about 25 mM, more preferably from about 15 mM to about 20 mM.
• the concentration of albumin in the albumin-comprising sample is from about 50 g/L to about 200 g/L, or from about 100 g/L to about 170 g/L, preferably from about 135 g/L to about 155 g/L, more preferably about 140 g/L to about 150 g/L, and wherein the sample is contacted with chelator at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the concentration of the chelating agent is from about 2.5 mM to about 100 mM, preferably from about 5 mM to about 50 mM, or between about 10 mM to about 25 mM, more preferably from about 15 mM to about 20 mM.
• the concentration of chelating agent is preferably less than 100 mM chelator, more preferably less than 50 mM chelator, or less than 25 mM chelator, or less than 15 mM chelator, or less than 5 mM chelator. Most preferably, the concentration of chelator is less than 1 mM or less than 500 pM, or less than 250 pM or less than 100 pM or 50 pM or less.
• the concentration of chelating agent is calculated relative to the concentration of copper in the albumin-comprising sample.
• the chelating agent is contacted with the albumin-comprising sample at an amount that corresponds to approximately at least a 2-fold higher concentration of chelator to copper, at least a 5-fold higher concentration of chelator to copper, at least 10-fold higher concentration of chelator to copper, at least 15-fold higher concentration of chelator to copper, at least 20-fold higher concentration of chelator to copper, at least a 25-fold higher concentration of chelator to copper, at least at least a 50-fold higher concentration of chelator to copper, at least a 75-fold higher concentration of chelator to copper, at least a 100-fold higher concentration of chelator to copper, at least a 125-fold higher concentration of chelator to copper, at least a 150- fold higher concentration of chelator to copper, at least a 175-fold higher concentration of chelator to copper, or at least
• the biodegradable chelating agent is (S,S)-EDDS, optionally the tri-sodium salt thereof, and wherein the (S,S)-EDDS is contacted with the albumin-comprising sample at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the (S,S)-EDDS is contacted with the albumincomprising sample at a concentration of from about 10 mM to about 20 mM, preferably about 15 mM.
• the biodegradable chelating agent is (S,S)-EDDS, optionally the tri-sodium salt thereof, and wherein the (S,S)-EDDS concentration is less than 100 mM, preferably less than 50 mM, or less than 25 mM, or less than 15 mM, or less than 5 mM. Most preferably, the concentration of (S,S)-EDDS is less than 1 mM or less than 500 pM, or less than 250 pM or less than 100 pM or 50 pM or less
• the concentration of (S,S)-EDDS to be used is calculated relative to the concentration of copper in the album in-comprising sample.
• the (S,S)-EDDS is contacted with the albumin-comprising sample at an amount that corresponds to approximately at least a 2-fold higher concentration of (S,S)-EDDS to copper, at least a 5-fold higher concentration of (S,S)- EDDS to copper, at least a 10-fold higher concentration of (S,S)-EDDS to copper, at least a 15-fold higher concentration of (S,S)-EDDS to copper, at least a 20-fold higher concentration of (S,S)-EDDS to copper, at least a 25-fold higher concentration of (S,S)-EDDS to copper or at least at least a 50-fold higher concentration of (S,S)-EDDS to copper, at least a 75-fold higher concentration of (S,S)-EDDS to copper, at least a 100- fold higher concentration of
• the concentration of albumin in the albumin-comprising sample is from about 40 g/L to about 250 g/L, and wherein the sample is contacted with (S,S)-EDDS (optionally the trisodium salt thereof) at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the concentration of (S,S)-EDDS (optionally the trisodium salt thereof is from about 2.5 mM to about 100 mM, preferably from about 5 mM to about 50 mM, or between about 10 mM to about 25 mM, more preferably from about 15 mM to about 20 mM.
• the concentration of albumin in the albumin-comprising sample is from about 50 g/L to about 200 g/L, or from about 100 g/L to about 170 g/L, preferably from about 135 g/L to about 155 g/L, more preferably about 140 g/L to about 150 g/L, and wherein the sample is contacted with (S,S)-EDDS (optionally the trisodium salt thereof), at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the concentration of (S,S)-EDDS is from about 2.5 mM to about 100 mM, preferably from about 5 mM to about 50 mM, or between about 10 mM to about 25 mM, more preferably from about 15 mM to about 20 mM.
• the chelating agent is EDTA, or a salt thereof, and wherein the EDTA is contacted with the albumin-comprising sample at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the EDTA is contacted with the albumin-comprising sample at a concentration of from about 10 mM to about 20 mM, preferably about 15 mM.
• the chelating agent is EDTA or a salt thereof, and wherein the EDTA concentration is less than 100 mM, preferably less than 50 mM, or less than 25 mM, or less than 15 mM, or less than 5 mM. Most preferably, the concentration of EDTA is less than 1 mM or less than 500 pM, or less than 250 pM or less than 100 pM or 50 pM or less
• the concentration of EDTA to be used is calculated relative to the concentration of copper in the albumin-comprising sample.
• the EDTA is contacted with the albumin-comprising sample at an amount that corresponds to approximately at least a 2-fold higher concentration of EDTA to copper, at least a 5-fold higher concentration of EDTA to copper, at least a 10- fold higher concentration of EDTA to copper, at least a 15-fold higher concentration of EDTA to copper, at least a 20-fold higher concentration of EDTA to copper, at least a 25-fold higher concentration of EDTA to copper or at least at least a 50-fold higher concentration of EDTA to copper, at least a 75-fold higher concentration of EDTA to copper, at least a 100-fold higher concentration of EDTA to copper, at least a 125-fold higher concentration of EDTA to copper, at least a 150-fold higher concentration of EDTA to copper, at least a 175-fold higher concentration of ED
• the concentration of albumin in the albumin-comprising sample is from about 40 g/L to about 250 g/L, and wherein sample is contacted with EDTA (or salt thereof) at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the concentration of EDTA is from about 2.5 mM to about 100 mM, preferably from about 5 mM to about 50 mM, or between about 10 mM to about 25 mM, more preferably from about 15 mM to about 20 mM.
• the concentration of albumin in the albumin-comprising sample is from about 50 g/L to about 200 g/L, or from about 100 g/L to about 170 g/L, preferably from about 135 g/L to about 155 g/L, more preferably about 140 g/L to about 150 g/L, and wherein sample is contacted with EDTA (or salt thereof) at a concentration of from about 10 pM to about 100 mM, preferably from about 25 pM to about 15 mM, more preferably from about 50 pM to about 1 mM, most preferably from about 50 pM to about 500 pM, especially about 50 pM.
• the concentration of EDTA is from about 2.5 mM to about 100 mM, preferably from about 5 mM to about 50 mM, or between about 10 mM to about 25 mM, more preferably from about 15 mM to about 20 mM.
• the chelating agent used, and amount thereof is sufficient to deplete the metal cations, preferably cupric ions, from the albumin-comprising sample to no more than about 5 pg metal cation/g protein, no more than 2.5 pg metal cation/g protein, no more than 2 pg metal cation/g protein, no more than 1 pg metal cation/g protein, preferably no more than about 0.8 pg/g protein.
• the chelator used and amount thereof preferably depletes the albumin-comprising fraction of copper such that the amount of copper remaining in the metal cation-depleted comprising fraction is no more than about 5 pg copper/g protein, no more than 2.5 pg copper/g protein, no more than about 2 pg copper/g protein, no more than 1 pg copper/g protein, preferably no more than about 0.8 pg copper /g protein, more preferably, no more than 0.5 pg copper/g protein.
• the chelating agent may be provided in the form of a chelating resin.
• the invention provides a process for preparing a purified albumin composition from an albumin-comprising sample obtained from blood-derived plasma, the process comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma; ii) contacting the albumin-comprising sample with a chelating resin for sequestering metal cations to obtain a metal cation-depleted album incomprising sample; thereby preparing a purified albumin composition.
• a process for preparing a purified albumin composition from an albumin-comprising sample obtained from blood-derived plasma comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma; ii) contacting the albumin-comprising sample with a chelating resin for sequestering metal cations under conditions that allow metal cations but not albumin to bind the resin, iii) recovering a metal cation-depleted albumin-comprising fraction; thereby preparing a purified albumin composition.
• the chelating resin is a cation exchange resin.
• the cation exchange resin may be a strong cation exchanger (i.e., strongly acidic) or a weak cation exchanger (i.e., weakly acidic).
• the cation exchange resin comprises acidic moieties including sulfonic acid, or carboxylic acid groups.
• the resin comprises a pore size that excludes or prevents albumin from contacting the negatively charged groups on the cationexchange resin (and therefore is such that the resin is not bound by albumin), particularly when the pH is below the isolelectric point of albumin (which is approximately pH 4.8).
• Non-limiting examples of suitable chelating resins for use in accordance with the invention include: Dowex® chelating resin A-1 , also known as Chelex 100, which is based on iminodiacetic acid in a styrene-divinylbenzene matrix.
• Other functional groups bound to chelating resins include aminophosphonic acids, thiourea, and 2-picolylamine.
• the cation resin may be AG® (or Dowex®) 50W cation exchange resin, having varying degrees of cross-linkage (e.g, AG® 50W-X2, AG® 50W-X4, AG® 50W-X8 and AG® 50W-X12, AG® 50W-X16 having 2%, 4%, 8% or 12% crosslinkage, respectively).
• the cation exchange resin may comprise sulfonic acid functional groups attached to a styrene divinylbenzene copolymer lattice.
• the contacting of the albumin-comprising sample with the ligand for binding to or for sequestering metal cations can be performed using a column-based chromatography method, or utilising a batch method.
• albumin-comprising sample of plasma used in any of the present methods may be any sample of plasma obtained from blood (or a blood derivative such as serum or plasma), and which contains albumin.
• blood derived plasma refers to the straw-coloured/pale yellow component of blood obtained from one or more blood donor(s). Methods of obtaining plasma from a donor will be apparent to a skilled person and/or described herein.
• blood derived plasma is obtained by removing red blood cells from donated blood.
• the blood derived plasma is obtained by plasmapheresis.
• the blood derived plasma is obtained by removing red blood cells from donated blood and/or plasmapheresis.
• the albumin-comprising sample may be any intermediate or derivative obtained from any plasma fractionation process.
• the albumin-comprising sample may be an intermediate obtained from a plasma salt-precipitation process.
• the albumin-comprising sample may be an intermediate or fraction obtained by alcohol fractionation of blood derived plasma.
• the albumin-comprising sample is a Cohn Fraction or a Kistler-Nitschmann Fraction or similar, obtained from cold-ethanol fractionation of blood derived plasma.
• the albumin-comprising sample is selected from the list consisting of Cryosupernatant, Cohn Supernatant I, Cohn Supernatant (l+)ll+lll, Filtrate A, Fraction IV filtrate, Fraction IVi filtrate, or Fraction IV4 filtrate.
• the albumin-comprising sample is Cohn Fraction V or Kistler-Nitschmann Precipitate C, or a suspension, filtrate or concentrate thereof.
• Filtrate D An example of a suspension, filtrate or concentrate of Precipitate C or of Fraction V may be referred to herein as Filtrate D.
• the albumin-comprising sample is a chromatographic purified fraction. Examples of such fractions are illustrated in Figure 1 .
• the pH of the albumin-comprising sample is between about 4.6 to about 4.8, particularly when the albumin-comprising sample is Cohn Fraction V or Kistler-Nitschmann Precipitate C.
• the pH of the albumin-comprising sample may be in the neutral range, preferably between about 6.8 to about 7.2, more preferably between about 7.0 to about 7.2, particularly when the albumin-comprising sample comprises a filtrate of chromatographic purified fraction of Cohn Fraction V or Kistler-Nitschmann Precipitate C.
• the process comprises resuspending a precipitate comprising an albumin-comprising sample of blood, prior to contacting the sample with the ligand for binding to or for sequestering metal cations.
• the resuspending is in water for injection (WFI).
• the metal cation-depleted albumin-comprising sample contains at least 10%, 20%, 30%, 40% or 50% less metal cations than the album in-comprising sample obtained from blood prior to contacting with a ligand for binding to or for sequestering metal cations.
• the level of metal cations in the metal cation-depleted albumin-comprising sample is reduced by at least about 10%, 20%, 30%, 40% or 50% compared to the albumin-comprising sample obtained from blood prior to contacting with a ligand for binding to or for sequestering metal cations.
• the methods may further comprises an optional step of acidification.
• the acidification may occur before or after the album in-comprising sample is contacted with the ligand for binding to or for sequestering metal cations.
• the ligand is a chelating agent
• the step of acidification is performed after the step of contacting the album incomprising sample with the chelating agent.
• the ligand is a cation-exchange resin
• the step of acidification is performed prior to contacting the album incomprising sample with the cation-exchange resin.
• Acidification is preferably performed by contacting the album in-comprising sample (or the albumin-comprising sample depleted of metal cations) with an inorganic acid, optionally selected from the group consisting of: sulphuric acid (H2SO4), citric acid (CeHsO?), hydrochloric acid (HCI), phosphoric acid (H3PO4), oxalic acid (C2H2O4) and formic acid (CH2O2).
• H2SO4 sulphuric acid
• citric acid CeHsO?
• hydrochloric acid H3PO4
• oxalic acid C2H2O4
• formic acid CH2O2
• acidification is performed using sulphuric acid or hydrochloric acid.
• the pH of the sample may typically be in the range of between about 4.6 to about 5.0 prior to the addition of chelating agent.
• an optional step of acidification may be performed after the addition of chelating agent, resulting in a reduction in the pH of the albumin-comprising sample to a pH of approximately 3.0 to 4.5, preferably to a pH of between about 3.5 to about 4.5, or between about 3.6 to 4.4, or between about 3.7 to about 4.3, or between about 3.8 to about 4.2, most preferably between about 3.9 to about 4.2.
• the step of acidification results in a reduction in the pH of the albumin-comprising sample to a pH of about 3.9, about 4.0, about 4.1 or about 4.2.
• the pH of the albumin-comprising sample that is depleted of metal cations is no lower than about 4.5, no lower than about 4.2 or no lower than about 4.0 following acidification.
• the pH of the sample may be in the neutral range prior to the addition of chelating agent.
• the process comprises an acidification step to adjust the pH to about 5.6 to about 6.0, more preferably to about 5.8 or about 5.9
• the process further comprises subjecting the albumin-comprising sample that is depleted of metal cations (or the acidified albumincomprising sample that is depleted of metal cations) to additional purification steps.
• the process therefore comprises subjecting the albumin-comprising sample that is depleted of metal cations (or the acidified albumin-comprising sample that is depleted of metal cations) to a pre-filtration step (e.g., clarifying depth filtration), ultrafiltration (e.g., diafiltration and/or concentration) and combinations thereof.
• a process for preparing a purified albumin pharmaceutical composition from an albumin-comprising sample obtained from blood-derived plasma comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is Cohn Fraction V or Kistler- Nitschmann Precipitate C, or a suspension, filtrate or concentrate thereof; ii) contacting the albumin-comprising sample with a biodegradable chelating agent to obtain a metal cation-depleted albumin-comprising sample, wherein the biodegradable chelating agent is preferably (S,S)-EDDS, or a salt thereof; iii) optionally, adjusting the pH of the sample prior to or immediately after the contacting with the chelating agent; iv) recovering the metal cation-depleted albumin-comprising sample, thereby preparing a purified albumin composition.
• the process further comprises a subsequent step of diafiltration to remove or substantially deplete any remaining chelating agent from the sample.
• the (S,S)-EDDS or salt thereof is added to a final concentration of at least about a 2-fold, at least about a 5-fold or at least about a 10-fold, at least about a 50-fold, at least about a 100-fold or at least about a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the (S,S)-EDDS is added to a concentration of from about 25 pM to about 20 mM, preferably from about 50 pM to about 15 mM, more preferably from about 50 pM to about 500 pM, most preferably about 50 pM.
• the invention relates to a process comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is Cohn Fraction V or Kistler- Nitschmann Precipitate C; ii) contacting the albumin-comprising sample with a biodegradable chelating agent to obtain a metal cation-depleted albumin-comprising sample, wherein the biodegradable chelating agent is preferably (S,S)-EDDS, or a salt thereof; iii) optionally, adjusting the pH of the sample after the contacting with the chelating agent, wherein preferably, the pH is adjusted to a pH of about 3.9 to about 4.3, more preferably, about 4.1 to 4.2; iv) recovering the metal cation-depleted albumin-comprising sample, thereby preparing a purified albumin composition.
• an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is Cohn Fr
• the process further comprises a subsequent step of depth filtration, and pH adjustment to a pH of about 7.0 to about 7.4, preferably about 7.2 to about 7.3, preferably about 7.2, followed by diafiltration to remove or substantially deplete any remaining chelating agent from the sample.
• the (S,S)-EDDS or salt thereof is added to a final concentration of at least about a 2-fold, at least about a 5-fold or at least about a 10-fold, at least about a 50-fold, at least about a 100-fold or at least about a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the (S,S)-EDDS is added to a concentration of from about 25 pM to about 20 mM, preferably from about 50 pM to about 15 mM, more preferably from about 50 pM to about 500 pM, most preferably about 50 pM.
• the invention relates to a process comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is a filtrate of Cohn Fraction V or Kistler-Nitschmann Precipitate C (eg Filtrate D); ii) optionally, adjusting the pH of the sample wherein preferably, the pH is adjusted to a pH of about 5.6 to 6.0, more preferably about 5.8 or about 5.9; iii) contacting the albumin-comprising sample with a biodegradable chelating agent to obtain a metal cation-depleted albumin-comprising sample, wherein the biodegradable chelating agent is preferably (S,S)-EDDS, or a salt thereof; iv) recovering the metal cation-depleted albumin-comprising sample, thereby preparing a purified albumin composition.
• an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is a
• the process further comprises a subsequent step of diafiltration to remove or substantially deplete any remaining chelating agent from the sample.
• the (S,S)-EDDS or salt thereof is added to a final concentration of at least about a 2-fold, at least about a 5-fold or at least about a 10-fold, at least about a 50-fold, at least about a 100-fold or at least about a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the (S,S)-EDDS is added to a concentration of from about 25 pM to about 20 mM, preferably from about 50 pM to about 15 mM, more preferably from about 50 pM to about 500 pM, most preferably about 50 pM.
• a process for preparing a purified albumin pharmaceutical composition from an albumin- comprising sample obtained from blood-derived plasma comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is Cohn Fraction V or Kistler- Nitschmann Precipitate C, or a suspension, filtrate or concentrate thereof; ii) contacting the albumin-comprising sample with a chelating agent to obtain a metal cation-depleted albumin-comprising sample, wherein the chelating agent is preferably EDTA, or a salt thereof; iii) optionally, adjusting the pH of the sample prior to or immediately after the contacting with the chelating agent; iv) recovering the metal cation-depleted albumin-comprising sample, thereby preparing a purified albumin composition.
• the process further comprises a subsequent step of diafiltration to remove or substantially deplete any remaining chelating agent from the sample.
• the EDTA or salt thereof is added to a final concentration of at least about a 2-fold, at least about a 5-fold or at least about a 10-fold, at least about a 50-fold, at least about a 100-fold or at least about a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the EDTA is added to a concentration of from about 25 pM to about 20 mM, preferably from about 50 pM to about 15 mM, more preferably from about 50 pM to about 500 pM, most preferably about 50 pM.
• the invention relates to a process comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is Cohn Fraction V or Kistler- Nitschmann Precipitate C; ii) contacting the albumin-comprising sample with a chelating agent to obtain a metal cation-depleted albumin-comprising sample, wherein the chelating agent is preferably EDTA, or a salt thereof; iii) optionally, adjusting the pH of the sample after the contacting with the chelating agent, wherein preferably, the pH is adjusted to a pH of about 3.9 to about 4.3, more preferably, about 4.1 to 4.2; iv) recovering the metal cation-depleted albumin-comprising sample, thereby preparing a purified albumin composition.
• the process further comprises a subsequent step of depth filtration, and pH adjustment to a pH of about 7.0 to about 7.4, preferably about 7.2 to about 7.3, preferably about 7.2, followed by diafiltration to remove or substantially deplete any remaining chelating agent from the sample.
• the EDTA or salt thereof is added to a final concentration of at least about a 2-fold, at least about a 5-fold or at least about a 10-fold, at least about a 50-fold, at least about a 100-fold or at least about a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the EDTA is added to a concentration of from about 25 pM to about 20 mM, preferably from about 50 pM to about 15 mM, more preferably from about 50 pM to about 500 pM, most preferably about 50 pM.
• the invention relates to a process comprising: i) providing an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is a filtrate of Cohn Fraction V or Kistler-Nitschmann Precipitate C (eg Filtrate D); ii) optionally, adjusting the pH of the sample wherein preferably, the pH is adjusted to a pH of about 5.6 to 6.0, more preferably about 5.8 or about 5.9; iii) contacting the albumin-comprising sample with a chelating agent to obtain a metal cation-depleted albumin-comprising sample, wherein the chelating agent is preferably EDTA, or a salt thereof; iv) recovering the metal cation-depleted albumin-comprising sample, thereby preparing a purified albumin composition.
• an albumin-comprising sample obtained from blood-derived plasma, preferably wherein the sample is a filtrate of Cohn Fraction V or Kistler-
• the process further comprises a subsequent step of diafiltration to remove or substantially deplete any remaining chelating agent from the sample.
• the EDTA or salt thereof is added to a final concentration of at least about a 2-fold, at least about a 5-fold or at least about a 10-fold, at least about a 50-fold, at least about a 100-fold or at least about a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the EDTA is added to a concentration of from about 25 pM to about 20 mM, preferably from about 50 pM to about 15 mM, more preferably from about 50 pM to about 500 pM, most preferably about 50 pM.
• the present invention also provides a purified albumin product comprising a concentration of copper of no more than about 2.0 pg/g protein, no more than about 1 .5 pg/g protein, no more than about 1 pg/g protein, no more than about 0.8 pg/g protein, no more than about 0.5 pg/g protein or no more than about 0.2 pg/g protein.
• the present invention also provides a purified albumin product obtained or obtainable by any process described herein.
• the purified albumin product comprises a concentration of copper of no more than about 2.0 pg/g albumin, no more than about 1.5 pg/g albumin, no more than about 1 pg/g albumin, no more than about 0.8 pg/g albumin or no more than about 0.6 pg/g albumin, no more than about 0.5 pg/g albumin, or no more than about 0.2 pg/g albumin.
• a purified albumin composition suitable for pharmaceutical use refers to a composition which meets the pharmacopoeia standards, such as Ph. Eur, or USP or as otherwise herein defined.
• a purified albumin composition suitable for pharmaceutical use is a composition that meets the pharmacopoeia standards, such as Ph. Eur, or USP, and which is specifically formulated for administration to humans.
• the purified albumin products described herein may be formulated into an hSA drug product or pharmaceutical composition and to contain appropriate amounts of albumin as the active ingredient according to pharmacopeia standards such as Ph. Eur. or USP.
• the following excipients may be included: sodium N-acetyl-tryptophan (stabiliser); sodium caprylate (stabiliser); sodium chloride (tonicity agent); water for injections (solvent).
• the protein content may be adjusted as required to manufacture 4%, 5%, 20% and 25% Human Albumin Solution (hSA).
• the final hSA formulations are selected from the group consisting of: i) Protein (4% w/v), sodium (140 mM) and caprylate (6.4 mM) for 4% w/v hSA; ii) Protein (5% w/v), sodium (140 mM) and caprylate (8 mM) for 5% w/v hSA; iii) Protein (20% w/v) and caprylate (32 mM) for 20% w/v hSA; iv) Protein (25% w/v) and caprylate (40 mM) for 25% w/v hSA.
• the final hSA formulations may be selected from the group consisting of: i) Protein (4% w/v), 3.2 mM sodium N-acetyltryptophanate and 3.2 mM sodium caprylate for 4% w/v hSA; ii) Protein (5% w/v), 4 mM sodium N- acetyltryptophanate and 4 mM sodium caprylate for 5% w/v hSA; iii) Protein (20% w/v) 0.016 M sodium N-acetyltryptophanate and 0.016 M sodium caprylate for 20% w/v hSA; iv) Protein (25% w/v) 0.02 M sodium N-acetyltryptophanate and 0.02 M sodium caprylate for 25% w/v hSA.
• the hSA formulations preferably do not comprise preservatives.
• an hSA pharmaceutical composition (drug product) will preferably meet the appropriate Pharmacopoeia standard.
• the hSA preparation is sterile; pyrogen free; has endotoxin levels below 0.5 III per mL for solutions less than 50 g/L, or less than 1.3 IU per mL for solutions from 50 g/L to 200 g/L, or less than 1 .7 lU/mL for solutions greater than 200 g/L; an aluminium content of a maximum of 200 pg/L, a prekallikrein activator (PKA) maximum of 35 lU/mL; a haem content not greater than 0.15; a potassium maximum of 0.05 mmol per gram of protein; a sodium maximum of 160 mmol/L and 95 % to 105% of the content of Na stated on the label; a maximum of 10% polymers and aggregates; not
• the album in-comprising sample is obtained from human blood.
• Figure 1 Schematic flow diagram comparing various methods for the preparation of plasma protein fraction and human albumin solution from blood derived plasma at industrial scale (from Br J Anaesth, Volume 85, Issue 6, 1 December 2000, Pages 887-895).
• Figure 2 Example of dark sediments found in preparations of albumin.
• FIG. 3 Copper concentration (pg/gram protein) after addition of 50 mM chelating agent to Filtrate D.
• Figure 4 Copper concentration (pg/gram protein) after addition of 50 mM chelating agent to Filtrate D, followed by pH adjustment to 5.8-5.9.
• Figure 5 Copper Concentration (mg/kg protein) following addition of various chelators (5-50 mM) to resuspended Precipitate C (Res C). Cont (control); (S,S)-EDDS at 15 mM, EDTA at 5 mM, IDS at 15 mM and 50 mM, NTA at 15 mM.
• FIG 6 Concentration of copper normalized to albumin concentration (pg copper/g albumin). Copper concentration in the paste resuspension, filtrate D and retentate (after LIF/DF), is shown relative to (S,S)-EDDS concentration in the resuspension (0 mM to 15 mM).
• Figure 7 Concentration of copper normalized to albumin concentration (pg copper/g albumin). Copper concentrated in the paste resuspension, filtrate D and retentate (after LIF/DF), is shown relative to (S,S)-EDDS concentration in the resuspension (0.05 mM to 1 mM).
• Figure 8 Copper reduction using acidification and Dowex cation exchange chromatography of Resuspended Precipitate C or Filtrate D.
• Figure 9 Accelerated stability study of albumin preparations obtained by various methods including acidification + cation exchange or addition of a chelator. Shown are albumin samples: not subjected to chelator treatment or cation exchange (control, at 8 month time point); subjected to acidification + cation exchange at 7 month time point (column and batch methods); subjected to treatment with EDTA (8 month time point); and subjected to treatment with (S,S)-EDDS (8 month time point).
• Plasma-protein products such as human albumin (hSA), immunoglobulin (IgG), clotting factor concentrates (clotting Factor VIII, clotting Factor IX, prothrombin complex etc.) and inhibitors (antithrombin, C1 -inhibitor etc.).
• hSA human albumin
• IgG immunoglobulin
• clotting factor concentrates clotting Factor VIII, clotting Factor IX, prothrombin complex etc.
• inhibitors antithrombin, C1 -inhibitor etc.
• hSA Human Proteins
• hSA is routinely administered in clinical settings when there are existing or anticipated clinical problems or complications from reduced oncotic pressure, and/or as an adjunct to diuretic therapy.
• the formation of dark sediments and aggregates have been observed in different preparations of serum albumin.
• the present inventors have identified improved processes for the purification of albumin from serum, including methods which minimise the development of such sediments and aggregates.
• the inventors believe that the dark sediments are caused by metal cations bound to albumin and to other proteins such as ceruloplasmin, which are often present in albumin preparations.
• the present invention is based on the findings by the inventors that dark sediments are likely caused by the presence of copper (in the form of CuS, copper (II) sulfide) in albumin preparations, with proteolytic activity of contaminating proteases also contributing to aggregation.
• the present invention therefore seeks to address some of the limitations of prior art methods for the preparation of serum albumin products.
• the methods of the present invention find application in removing contaminating metal cations from any albumin-comprising sample of blood.
• the starting material for use in any of the methods described herein can be any plasma-derived composition that comprises albumin.
• Figure 1 herein, provides an overview of various fractionation methods for obtaining pharmaceutical compositions of albumin from blood derived plasma at an industrial scale.
• the plasma or serum is usually subjected to alcohol fractionation, which may be combined with other purification techniques like chromatography, adsorption or precipitation.
• alcohol fractionation may be combined with other purification techniques like chromatography, adsorption or precipitation.
• other processes can also be used.
• the album in-comprising precipitate can be precipitate V obtained according to the Cohn’s methods such as the Method 6, Cohn et. al. J. Am; Chem. Soc., 68 (3), 459-475 (1946), the Method 9, Oncley et al. J. Am; Chem.
• Normal plasma “hyperimmune plasma” (such as hyperimmune anti-D, tetanus or hepatitis B plasma) or any plasma equivalent thereto can be used as a starting material in the cold ethanol fractionation processes described herein.
• hyperimmune plasma such as hyperimmune anti-D, tetanus or hepatitis B plasma
• any plasma equivalent thereto can be used as a starting material in the cold ethanol fractionation processes described herein.
• the albumin-comprising composition or sample may be a cryosupernatant.
• cryosupernatant also called cryo-poor plasma, cryoprecipitate depleted plasma and similar
• plasma derived from either whole blood donations or plasmapheresis
• Cryoprecipitation is the first step in most plasma protein fractionation methods in use today, for the large- scale production of plasma protein therapeutics. The method generally involves pooling frozen plasma that is thawed under controlled conditions (e.g. at or below 6 °C) and the precipitate is then collected by either filtration or centrifugation.
• cryosupernatant The supernatant fraction, known to those skilled in the art as a "cryosupernatant", is generally retained for use.
• the resulting cryo-poor plasma has reduced levels of Factor VIII (FVIII), von Willebrand factor (VWF), Factor XIII (FXIII), fibronectin and fibrinogen. While the levels of FVIII are greatly reduced, levels of fibrinogen can be as much as 70 % of original levels.
• Cryosupernatant provides a common feedstock used to manufacture a range of therapeutic proteins, including albumin.
• the albumin-comprising composition or sample may be an ethanol precipitate.
• the supernatant of the 8 % ethanol-precipitate (method of Cohn et al.; Schultze et al. (see above), p. 251 ), precipitate V (method of Oncley et al.; Schultze et al. (see above) p. 253) or precipitate C (method of Kistler and Nitschmann; Schultze et al. (see Schultze above), p. 253) are examples of a source of albumin compatible with industrial scale plasma fractionation.
• the source of albumin may also be any filtrate, concentrate or suspension of precipitate V or precipitate C (such as, for example, Filtrate D which is obtained from subjecting precipitate C to clarifying depth filtration).
• the total protein concentration of the starting material is between about 10-350 g/L, about 50-300 g/L, about 60 to about 250 g/L, about 75-150 g/L, most preferably about 50-100 g/L.
• the ligand for binding to or for sequestering metal cations in the albumin-comprising sample is a chelating agent.
• the chelating agent is used to deplete the albumin-comprising sample of copper ions, although it will be appreciated that the chelating agent can be used to deplete the protein fraction of other metal cations that may contribute to the formation of sediments in the final composition.
• any suitable chelating agent may be used in order to deplete the albumin-comprising sample of metal cations.
• the chelating agent binds to a divalent metal cation.
• the chelating agent binds to copper (II) ions, wherein the copper is optionally present in the albumin solution as copper (II) sulfide.
• Non-limiting examples of suitable chelators for use in the methods of the present invention include: ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(
• EDTA ethylenediaminet
• the chelating agent is selected from the group consisting of: ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycinediacetic acid (MGDA), and nitrilotriacetic acid (NTA), most preferably EDDS (especially (S,S)-EDDS), or a salt thereof, including a calcium or sodium salt thereof.
• EDDS ethylenediamine-N,N'-disuccinic acid
• IDS iminodisuccinic acid
• MGDA methylglycinediacetic acid
• NDA nitrilotriacetic acid
• EDTA ethylenediaminetetraacetic acid
• 2, 2', 2", 2'"- (Ethane-1 ,2-diyldinitrilo)tetraacetic acid N,N'-Ethane-1 ,2-diylbis[N-
• EDTA (carboxymethyl)glycine], diaminoethane-tetraacetic acid and edetic acid; CAS no: 60- 00-4 ), is a hexadendate ligand that binds to metal ions including Cu 2+ , Ca 2+ , Mg 2+ and Fe 3+ .
• EDTA may be provided in free acid form, or as a salt, such as dehydrate disodium.
• ethylene glycol-bis([3-aminoethyl ether)-N,N,N',N'-tetraacetic acid is a hexadendate ligand that binds to metal ions including Cu 2+ , Ca 2+ , Mg 2+ and Fe 3+ .
• ethylenediamine-N,N'-disuccinic acid is an aminopolycarboxylic acid. It is a colourless solid that is used as chelating agent that may offer a biodegradable alternative to EDTA, which is currently used on a large scale in numerous applications.
• EDDS has two chiral centers, and as such three stereoisomers. These are the enantiomeric (R,R) and (S,S) isomers and the achiral meso (R,S) isomer.
• the (S,S) stereoisomer is preferred. Therefore in a preferred embodiment the chelating agent is (S,S)-EDDS.
• the chelating agent is the trisodium salt of (S,S)- EDDS.
• IDS refers to iminodisuccinic acid, or a salt thereof, such as tetrasodium iminidisuccinate (also referred to as N-(1 ,2-dicarboxyethyl)aspartic acid; CAS no: 144538-83-0).
• Tetrasodium iminodisuccinate is a chelating agent, forming complexes of moderate stability which includes (as a pentadentate ligand) alkaline earth and polyvalent heavy metal ions with one molecule of water in an octahedral structure.
• Tetrasodium iminodisuccinate is classified as readily biodegradable according to OECD methods.
• methylglycinediacetic acid usually provided in salt form: trisodium N-(1-carboxylatoethyl)iminodiacetate, methylglycinediacetic acid trisodium salt (MGDA-Na3) or trisodium a-DL-alanine diacetate (a-ADA), is the trisodium anion of N- (l -carboxyethyl)iminodiacetic acid and a tetradentate complexing agent. It forms stable 1 :1 chelate complexes with cations having a charge number of at least +2, e.g. the "hard water forming" cations Ca 2+ or Mg 2+ .
• TETA triethylenetetramine
• INN trientine
• iminodiacetic acid is a dicarboxylic acid amine, of which the dianion is a tridentate ligand, forming metal complexes by forming two, fused, five membered chelate rings.
• the proton on the nitrogen atom can be replaced by a carbon atom of a polymer to create an ion-exchange resin, such as chelex 100.
• NTA nitrilotriacetic acid
• Triglycine also known as N,N- Bis(carboxymethyl)glycine 2-[Bis(carboxymethyl)amino]acetic acid; Triglycine, CAS no: 139-13-9
• NTA is a colourless solid that is used as a chelating agent, which forms coordination compounds with metal ions (chelates) such as Ca 2+ , Co 2+ , Cu 2+ , and Fe 3+ .
• NTA is a tripodal tetradentate trianionic ligand.
• tripolyphosphate also known as sodium triphosphate (STP), sodium tripolyphosphate (STPP), or Pentasodium triphosphate (CAS no: 7758- 29-4) is an inorganic compound with formula NasPsO . It is the sodium salt of the polyphosphate penta-anion, which is the conjugate base of triphosphoric acid. It is produced on a large scale as a component of many domestic and industrial products, especially detergents. It binds strongly to metal cations as both a bidentate and tridentate chelating agent.
• diethylenetriaminepentaacetic acid DTPA
• pentetic acid CAS no: 67-43-6
• DTPA diethylenetriaminepentaacetic acid
• CAS no: 67-43-6 pentetic acid
• the molecule can be viewed as an expanded version of EDTA and is used similarly. It is a white solid with limited solubility in water.
• DDC sodium diethyldithiocarbamate
• CAS no: 148-18-5 Sodium diethylcarbamodithioate
• coorindates to many "softer" metals via two sulfur atoms.
• Other more complicated bonding modes are known including binding as unidentate ligand and a bridging ligand using one or both sulfur atoms.
• L-Glutamic acid A/,A/-diacetic acid, tetrasodium salt is also known as tetrasodium glutamate diacetate (CAS no: 51981 -21-6).
• the chelator may be provided in salt form, including a calcium, hydrochloride, or mono-, di-, tri- tetra-sodium salt thereof.
• the chelator is combined with the albumin-comprising sample at a concentration of at least about a 2-fold, at least about a 5-fold, at least about a 10-fold, at least about a 25-fold, at least about a 50-fold, at least about a 100- fold or at least about a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the chelator is added to a concentration of between about 25 pM to about 20 mM, preferably between about 50 pM to about 15 mM, more preferably between about 50 pM to about 500 pM, most preferably about 50 pM.
• the biodegradable chelating agent is (S,S)-EDDS
• the (S,S)-EDDS is combined with the albumin-comprising sample at a concentration of at least about a 2-fold, or at least about a 5-fold, at least about a 10- fold, at least about a 25-fold, at least about a 50-fold, at least about a 100-fold or at least about a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the (S,S)-EDDS is added to a concentration of between about 25 pM to about 20 mM, preferably between about 50 pM to about 15 mM, more preferably between about 50 pM to about 500 pM, most preferably about 50 pM.
• the chelator is combined with the albumin-comprising sample at a concentration of at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 25-fold, at least a 50-fold, at least a 100-fold or at least a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the chelator is added to a concentration of between 25 pM to 20 mM, preferably between 50 pM to 15 mM, more preferably between 50 pM to 500 pM, most preferably 50 pM.
• the biodegradable chelating agent is (S,S)-EDDS
• the (S,S)-EDDS is combined with the albumin-comprising sample at a concentration of at least at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 25-fold, at least a 50-fold, at least a 100-fold or at least a 200-fold higher concentration than the concentration of copper in the sample; optionally wherein the (S,S)-EDDS is added to a concentration of between 25 pM to 20 mM, preferably between 50 pM to 15 mM, more preferably between 50 pM to 500 pM, most preferably 50 pM.
• Charged molecules such as proteins, peptides, and/or amino acids, as well as other solutes, may be separated from compositions by means of ion exchange.
• Ion exchange materials such as ion exchange resins, typically comprise two types of ions: ions bound within or on the resin, and an oppositely charged counterion.
• charged molecules in the composition may displace the counterions and bind to the resin.
• the charged molecules bound to the resin may be competitively and sequentially displaced or eluted from the resin in an order that is inversely related to the binding affinity of the charged molecules by raising the concentration of counterions in the sample.
• ion exchange see Moore, et al., “Chromatography of amino acids on sulfonated polystyrene resins,” 1958, Analytical Chemistry, 30:1185-1190.
• Strong cation exchange resins/media are strongly acidic, and generally contain fully ionized acidic groups such as sulfonic acid groups or the corresponding salts. These exchangers are negatively charged, and bind cations very strongly.
• the exchange capacity of strong acid or base resins is typically independent of the pH of the sample contacting the resin.
• strong cation exchangers include: sulfonic acid, Trisacryl, sulphopropyl, and the like.
• Weak cation exchange resins/media contain weak acids, such as carboxylic acid groups or the corresponding salts (e.g., carboxymethyl (CM) cellulose, Chelex-100, etc.). Since the degree of dissociation of a weak acid resin is influenced by pH, resin capacity depends in part on solution pH. For example, a typical weak acid resin has very limited capacity below a pH of 6.0. A weak cation exchanger therefore operates across a narrow pH range (between about 6 and 7). Thus, by “weak cation exchange resin” or “weak cation exchange material” it is meant that the exchanger is a weak acid that will be negatively charged when the pH is above the pK a of the exchanger. Examples of weak cation exchangers include carboxymethyl (CM), phosphono and polyaspartic acid and the like.
• Ion exchange resins may comprise a variety of charged groups.
• cation exchange resin may comprise charged groups such as carboxymethyl (CM), sulfopropyl (SP), and/or methyl sulfonate (S). These charged groups may be attached to a variety of core materials including those that are agarose-based (e.g., SEPHAROSE CL-6B, SEPHAROSE FAST FLOW, and SEPHAROSE HIGH PERFORMANCE), cellulose-based (e.g., DEAE SEPHACEL), dextran-based (e.g., SEPHADEX), silica-based, and synthetic polymer based.
• agarose-based e.g., SEPHAROSE CL-6B, SEPHAROSE FAST FLOW, and SEPHAROSE HIGH PERFORMANCE
• cellulose-based e.g., DEAE SEPHACEL
• dextran-based e.g., SEPHADEX
• silica-based e.g., silica-based,
• the ion exchangers are strong cation exchangers (i.e., strongly acidic resins).
• the cation exchange resin used herein preferably comprises a sulfonic cation exchange resin, for example comprising sulfonic acid (-SO3H) bound to matrix styrene divinylbenzene.
• the cation exchange resin may comprise an iminodiacetatic acid chelating cation exchange resin.
• the cation exchange material used in the methods described herein may be in various formats.
• the cation exchanger may be coated on beads, may be in the form of a membrane ion exchange resin, may be coated onto the interior surface or a portion of the interior surface of the container in which the separation is being performed, and/or may be coated on an object, such as a magnetic stir bar or rod, present in the container in which the separation is being performed.
• the cation exchange material may be in the form of a strong acid cation exchange resin (H-form), or chelating ion exchange resin (amino phosphate) in a polyethylene matrix.
• H-form strong acid cation exchange resin
• amino phosphate amino phosphate
• examples of such matrices include 3M® Metal Ion Purifiers filter cartridges and discs.
• Still further embodiments contemplate the use of an iminodiacetate functionalised poly[styrenedivinylbenzenel) resin, for example, Empore® (3M®) extraction discs.
• Empore® Empore®
• the cation exchange resin comprises a pore size that excludes albumin from contacting the negatively charged cation-exchange groups/ligands of the resin (such that preferably, the resin does not bind the albumin present in the album incomprising solution, particularly when the pH is below the isolelectric point of albumin (which is approximately pH 4.8).
• the cation exchange resin is a fine mesh resin for use in fine chemical and pharmaceutical column preparations.
• the resin used herein is capable of free- flowing movement relative to the rest of the dispersion.
• the resin particles are preferably capable of independent movement relative both to other resin particles and relative to the other dispersion components.
• the aqueous dispersions comprising the free-flowing resin may therefore be amenable to agitation, in contrast to the relatively immobile state of resin that is packed in a column.
• the ligand for binding to or for sequestering metal cations may comprise a cation exchange resin.
• suitable resins include cation exchange resins comprising a sulfonic cation exchange resin, for example comprising sulfonic acid (-SO3H) bound to matrix styrene divinylbenzene.
• sulfonic acid sulfonic acid
• Commercially available examples of such resins are known to the skilled person and include the AG® or Dowex® fine mesh spherical strong acid cation exchange resins, especially the 50W series, and having varying degrees of resin cross-linking.
• the resin may be 50W X2, 50W X4, 50W X8, 50WX12, or 50WX16, having 2%, 4%, 8%, 12% or 16%, of divinylbenzene in the resin co-polymer, respectively.
• chelating resins include resins with an iminodiacetate functional group, such as Amberlite IRC 748, Lewatit TP 207, Lewatit TP 208, Purolite S 930, Lewatit MonoPlus TP 207, iminodiacetic acid fiber lonex IDA-Na, and Dowex M 4195.
• resins with an iminodiacetate functional group such as Amberlite IRC 748, Lewatit TP 207, Lewatit TP 208, Purolite S 930, Lewatit MonoPlus TP 207, iminodiacetic acid fiber lonex IDA-Na, and Dowex M 4195.
• Other suitable resins will be apparent to those skilled in the art, and are described, for example, in Edebali and Pehlivan (2016) Powder Technology, 301 : 520- 525, incorporated herein by reference.
• the methods of the invention may also comprise a step of acidification (a pH shift) to further improve the purity of the albumin-comprising sample and to minimise the impact of contaminating proteases.
• a step of acidification a pH shift
• the step of acidification may be prior to or after the contacting of the albumin-comprising sample with the chelating agent.
• the chelator is added to the sample first, followed by the step of acidification.
• a filtrate of Precipitate C or Fraction V is used (e.g., Filtrate D)
• the step of acidification may be performed first, followed by addition of the chelating agent.
• the step of acidification comprises contacting the album in-comprising sample (or the albumin-comprising sample depleted of metal cations) with an inorganic acid, optionally selected from the group consisting of: sulphuric acid (H2SO4), citric acid (CeHsO?), hydrochloric acid (HCI), phosphoric acid (H3PO4), oxalic acid (C2H2O4) and formic acid (CH2O2).
• H2SO4 sulphuric acid
• citric acid CeHsO?
• hydrochloric acid H3PO4
• oxalic acid C2H2O4
• formic acid CH2O2
• acidification is performed using sulphuric acid or hydrochloric acid.
• the step of acidification results in a reduction in the pH of the album in-comprising sample to a pH of approximately 3.0 to 4.8, preferably to a pH of between about 4.0 to about 4.8, or between about 4.0 to 4.4, most preferably between about 3.9 to about 4.2.
• the step of acidification results in a reduction in the pH of the album incomprising sample to a pH of about 3.8, about 3.9, about 4.0, about 4.1 , about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7 or about 3.8, especially to a pH of about 3.9, about 4.0, about 4.1 or about 4.2.
• the step of acidification results in a reduction in the pH of the albumincomprising sample to a pH of approximately 5.6 to 6.0, preferably to a pH of between about 5.8 to about 5.9.
• acidification optionally results in a reduction of the pH of the albumin-comprising sample to a pH of between about 4.6 to about 4.8.
• the inorganic acid is added to the albumin-comprising sample at a concentration of between about 0.01 M to about 5.0 M (molar), of between about 0.1 M to about 2.5 M, of between about 0.2 M to about 2.0 M, of between about 0.2 M to about 1 .0 M, more preferably at a concentration of between about 0.25 M to about 0.5 M.
• the inorganic acid is sulphuric acid or hydrochloric acid, and is added to the albumin-comprising sample at a concentration of between about 0.01 M to about 5.0 M (molar), of between about 0.1 M to about 2.5 M, of between about 0.2 M to about 2.0 M, of between about 0.2 M to about 1 .0 M, more preferably at a concentration of between about 0.25 M to about 0.5 M.
• any chelating agent present in the albumin-comprising fraction should preferably be removed prior to formulation of an hSA pharmaceutical composition. Accordingly, in preferred embodiments, the present invention provides for various addition filtration methods following the step of recovering a metal cation-depleted album in-comprising fraction according to any aspect of the invention.
• the subsequent steps of filtration may include a diafiltration step to remove any chelating agent, optionally including a concentration step prior to and/or following diafiltration.
• a diafiltration step to remove any chelating agent, optionally including a concentration step prior to and/or following diafiltration.
• the skilled person will be familiar with suitable diafiltration methods for removal of undesirable components of a protein product. For example, the skilled person will be able to determine the number of rounds of diafiltration required (e.g, volumes of diafiltration) to remove the chelating agent.
• a diafiltration step may also be useful for removal of other contaminants including aluminium, other low molecular salts and ethanol.
• the final albumin product is preferably also sterile filtered prior to dispensing and pasteurisation.
• the final hSA formulations are selected from the group consisting of: i) Protein (4% w/v), sodium (140 mM) and caprylate (6.4 mM) for 4% w/v hSA; ii) Protein (5% w/v), sodium (140 mM) and caprylate (8 mM) for 5% w/v hSA; iii) Protein (20% w/v) and caprylate (32 mM) for 20% w/v hSA; iv) Protein (25% w/v) and caprylate (40 mM) for 25% w/v hSA.
• the final hSA formulations may be selected from the group consisting of: i) Protein (4% w/v), 3.2 mM sodium N-acetyltryptophanate and 3.2 mM sodium caprylate for 4% w/v hSA; ii) Protein (5% w/v), 4 mM sodium N- acetyltryptophanate and 4 mM sodium caprylate for 5% w/v hSA; iii) Protein (20% w/v) 0.016 M sodium N-acetyltryptophanate and 0.016 M sodium caprylate for 20% w/v hSA; iv) Protein (25% w/v) 0.02 M sodium N-acetyltryptophanate and 0.02 M sodium caprylate for 25% w/v hSA.
• the hSA formulations preferably do not comprise preservatives.
• an hSA pharmaceutical composition according to the present invention will preferably meet the the appropriate Pharmacopoeia standard.
• the hSA preparation is sterile; pyrogen free; has endotoxin levels below 0.5 III per mL for solutions less than 50g/L, or less than 1.3 IU per mL for solutions from 50g/L to 200g/L, or less than 1 .7 lU/mL for solutions greater than 200g/L; an aluminium content of a maximum of 200 pg/L, a prekallikrein activator (PKA) maximum of 35 ILI/mL; a haem content not greater than 0.15; a potassium maximum of 0.05 mmol per gram of protein; a sodium maximum of 160 mmol/L and 95 % to 105% of the content of Na stated on the label; a maximum of 10% polymers and aggregates; not more than 5% of protein has a
• the temperature at which the methods of the present invention are performed will be a temperature which maintains the integrity of the albumin product.
• the methods are performed at a temperature of between 0°C to about 20°C, preferably between 0°C to about 10°C, more preferably between 2°C to about 8°C, most preferably between 2°C to about 4°C.
• Example 1 Characterisation of dark sediment in albumin products
• Example 2 Use of a chelating step to reduce copper levels in albumin preparations
• An album in-containing fraction was obtained from blood plasma according to the Kistler Nitschmann methods. Briefly:
• Frozen plasma was thawed prior to adjusting the pH of the plasma to pH 5.7 to 6.0 with acetate acetic acid buffer (pH 4) in a stainless steel tank. Ethanol 96% was added continuously to a concentration of 19% (V/V) while stirring and cooling to a temperature of - 5.5°C ⁇ 1.0°C. The pH was then adjusted to 5.70 to 5.90.
• the suspended precipitate A was removed by filtration after addition of perlite (filter aid) on polypropylene sheets. The filtrate of Precipitate A was collected in another stainless steel tank for further processing to albumin. The temperature was reduced to 7 ⁇ 0.5°C, and simultaneously 96% ethanol was added to a final concentration of 40% (V/V). The pH of this solution was adjusted, if necessary, to 5.95 to 6.00. Fraction IV precipitates and was then, by the use of filter aids (perlite and diatomaceous earth), filtered through a filter matrix support in order to remove the precipitate.
• filter aids perlite and
• filtrate IV was adjusted by 1.1 M acetic acid to 4.8 ⁇ 0.1 at a constant ethanol concentration of 40% and under cooling to a temperature of 7 ⁇ 1 °C.
• Precipitate C precipitates and was then separated from the ethanol solution by filtration with diatomaceous earth.
• Precipitate C contains almost exclusively albumin and filter aids.
• Precipitate C from one or more fractionation lots was first resuspended in water for injection (1 kg paste + 1 .7 kg water for injection) and then filtered at pH 4.7 ⁇ 0.1 through asbestos free depth filters to obtain Filtrate D.
• the pH of Filtrate D was adjusted to 7.2 ⁇ 0.1 by means of 1 M NaOH.
• chelators were selected for an initial series of experiments to determine if addition of chelating agent to an albumin-comprising plasma derivative, would be useful for depleting copper ions.
• an albumin-containing sample was obtained according to the method described in Example 2 to obtain Filtrate D. Aliquots of Filtrate D were taken, and the pH adjusted to between 4.0 and 7.2. Samples of the aliquots were then subjected to filtration through a 10 kDa membrane (Amicon) and centrifugation at 2000 rpm for 110 minutes.
• a positive control was also performed, using a solution of copper sulfate pentahydrate containing 0.5 mg/L of copper, which was also applied to a 10kDa membrane and centrifuged at 2000 rpm.
• the retentate and permeate following centrifugation were analysed to assess total protein concentration (g/L) and copper concentration (mg/kg).
• Example 4 Use of chelators in scale-up manufacturing of albumin product
• the chelators were added to resuspended Precipitate C or Precipitate V, at commercial scale (ie in a large tank).
• the pH was adjusted to about 4.0 - 4.8 with 1 M HCI as needed.
• the chelating agents were added at a concentration of 15 mM (5 mM for EDTA), because the results of Example 2 indicated that it is likely that a reduced amount of the chelating agent is required at a lower pH value.
• Example 5 Use of alternative concentrations of chelator in preparation of albumin product
• (S,S)-EDDS (as a solution of NasEDDS) was added to resuspended Precipitate C (obtained from a KN fractionation process) and the pH was adjusted to about 4.6 - 4.8 with 1 M HCI as needed. The mixture was then processed through depth filtration and ultrafiltration steps to obtain Filtrate D.
• the pH of Filtrate D was readjusted to pH 7.2-7.3 prior to ultrafiltration and then concentrated to 135 ⁇ 5 g protein/kg, and the subjected to diafiltration.
• the concentration of (S,S)-EDDS did not impact on the proteolytic activity in the end product, with all results being equal to or lower than 3 nkat/L.
• albumin e.g. in the monomer and aggregate content
• (S,S)-EDDS (as a solution of NasEDDS) was added to resuspended Precipitate C (obtained from a KN fractionation process) and the pH was adjusted to about 4.6 - 4.8 with 1 M HCI as needed. The mixture was then processed through depth filtration and ultrafiltration steps to obtain Filtrate D.
• the pH of Filtrate D was readjusted to pH 7.2-7.3 prior to ultrafiltration and then concentrated to 135 ⁇ 5 g protein/kg, and the subjected to diafiltration.
• the extent of copper depletion ranged from 73% (using 1 mM (S,S)-EDDS) to 82% (using 0.05 mM (S,S)-EDDS)
• the concentration of (S,S)-EDDS did not impact on the proteolytic activity in the end product, with all results being equal to or lower than 3 nkat/L.
• albumin e.g. in the monomer and aggregate content
• the albumin-comprising samples are subjected to downstream manufacturing processes.
• the staring material is Precipitate C
• the album in-containing sample is subjected to clarifying depth filtration as described in Example 2, to obtain metal-cation depleted Filtrate D.
• the neutralised Filtrate D solution is concentrated to about 130 to 140 g/kg albumin by ultrafiltration, then diafiltered first with at least five times the actual volume of 0.1 to 0.3 M sodium chloride, then with at least 2.5 times the amount of water for injection.
• the chelator along with any bound metal cations is removed. Also removed is aluminium, other low molecular salts and ethanol.
• the albumin solution is then formulated. This is achieved by adjusting the sodium content by adding sodium chloride to a concentration of 140 mmol/L. Then the stabilisers sodium caprylate and sodium N-acetyltryptophanate are added to the albumin solution, corresponding to 20 mmol/L for 25% albumin, 16 mmol/L for 20% albumin and 4 mmol/L for 5% albumin. Water for injection is added to dilute the albumin solutions to the target protein concentration and the pH is adjusted if required by hydrochloric acid or sodium hydroxide solution, respectively.
• the final albumin product is sterile filtered prior to being dispensed and then pasteurised.

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